4703 incagn1949, an anti-ox40 antibody with an optimal...
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IL-2 SecretionOX40 is a T cell co-stimulatory receptor that can enhance the magnitude and durability of T cell immune responses.Anti-OX40 agonist antibodies have shown significant single agent tumoricidal activity in preclinical models, and cancombine effectively with other immunomodulatory antibodies, targeted therapies and vaccines. OX40 agonists are able tocounteract the immunosuppressive tumor microenvironment and promote tumor-specific cellular immunity via at least twodistinct mechanisms: 1) promoting OX40 forward signaling in tumor-specific T cells; and 2) co-engaging Fc receptorsexpressed by tumor-associated effector cells, and facilitating the selective elimination of OX40high intratumoral regulatoryT cells.
INCAGN1949, an anti-OX40 human IgG1 antibody, was selected based on its ability to optimally enhance T cellresponsiveness under conditions of suboptimal T cell receptor stimulation. INCAGN1949 was shown to mediate effectiveapical OX40 clustering that is translated into effective downstream activation of the NFkB pathway. Notably, INCAGN1949was shown to maintain a sigmoidal dose response curve across a broad range of antibody concentrations. This suggests awide therapeutic window and may be advantageous for dosing considerations. By contrast, evaluation of reference OX40antibodies indicated an inverted U-shaped dose response curve, leading to impaired T cell responses at highconcentrations. INCAGN1949 was selected for clinical development based on its optimal agonist profile, further reinforcedby its ability to combine with other co-inhibitory and co-stimulatory antibodies to augment T cell responsiveness. Prior tohuman testing, the pharmacology and tolerability of INCAGN1949 was evaluated in non-human primates (NHPs).Pharmacokinetic (PK) and pharmacodynamic (PD) parameters were evaluated including longitudinal measurements ofserum cytokines, immune cell populations, activation state and T cell-mediated immune responses to reporter vaccineantigens. INCAGN1949 exhibited a linear PK profile and was well tolerated at all doses tested, with no maximum tolerateddose established. Co-administration of INCAGN1949 and vaccines in NHPs showed an immune-based PD signatureacross a broad exposure range. These studies were in line with in vitro findings and support a wide PD range forINCAGN1949 in patients. An important secondary mechanism of INCAGN1949 is the ability of its IgG1 Fc region tomediate selective depletion of OX40high intratumoral regulatory T cells. Immunohistochemistry and flow cytometry analysessupport the validity of this regulatory T cell depletion mechanism in a range of tumors.
The functional in vitro and in vivo attributes of INCAGN1949 make it suitable for clinical development. It is currently underevaluation in a Phase 1/2 study in subjects with advanced or metastatic tumors (NCT02923349).
INCAGN1949, an Anti-OX40 Antibody With an Optimal Agonistic Profile and the Ability to Selectively Deplete Intratumoral Regulatory T Cells
Author DisclosuresAna Gonzalez, Mariana Manrique, Ekaterina Breous, David Savitsky, Jeremy Waight, Lukas Swiech, Thomas Horn, Christopher Clarke, Yuqi Liu, Shiwen Lin, Jennifer Buell, Robert Stein, Marc van Dijk, Nicholas S. Wilson: Agenus Inc.: Employment and Stock Ownership. Jennifer Pulini, Kevin Heller, Reid Huber, Peggy Scherle, Gregory Hollis: Incyte Corporation:Employment and Stock Ownership. Taha Merghoub, Daniel Hirschhorn-Cymerman, Gerd Ritter, David Schaer: None.
AcknowledgmentsThe authors would like to thank Joseph Connolly and Zhenyu Li for their help characterizing and producing INCAGN1949.
Layout and printing support was provided by Evidence Scientific Solutions, Philadelphia, PA, funded by Incyte Corporation.
Abstract
4703Ana Gonzalez,1 Mariana Manrique,1 Lukasz Swiech,1 Thomas Horn,1 Ekaterina Breous,1,2 Jeremy Waight,1 David Savitsky,1 Yuqi Liu,1 Shiwen Lin,1 Christopher Clarke,1 Taha Merghoub,3 Daniel Hirschhorn-Cymerman,3 David Schaer,3 Gerd Ritter,4Jennifer Pulini,5 Kevin Heller,5 Peggy Scherle,5 Gregory Hollis,5 Reid Huber,5 Marc van Dijk,1,2 Jennifer Buell,1 Robert Stein,1 and Nicholas S. Wilson1Presented at the
American Association for Cancer Research Annual Meeting 2017Washington, DC, USA • April 1–5, 2017
Mechanism 2: Intratumoral Depletion of Treg Cells
• INCAGN1949 shows an optimal agonist profile across a broad dose range, which is mediated by its ability to engage and cluster OX40 on T cells
• INCAGN1949 cooperates with other immunomodulatory antibodies (both agonist and antagonist) to enhance T cell responsiveness
• Primary human tumors contain populations of FOXP3-expressing OX40high regulatory T cells, with the potential to be selectively depleted by the IgG1 Fc region of INCAGN1949
• INCAGN1949 has a linear PK profile in non-human primates and was well tolerated • INCAGN1949 co-administered with reporter vaccines in non-human primates promotes
enhanced T cell–mediated B cell immune responses• INCAGN1949 is an effective agonist of the OX40 pathway and has confirmed
immunomodulatory activity in vivo • INCAGN1949 is currently in Phase 1 clinical development (NCT02923349)
Summary
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Mechanism 1: OX40 Forward Signaling in Activated T Cells
References1. Croft M. Nat Rev Immunol. 2003;3:609–620.2. Bulliard Y, et al. Immunol Cell Biol. 2013;92:475–480.3. Croft M. Annu Rev Immunol. 2010;28:57–78.4. Voo KS, et al. J Immunol. 2013;191:3641–3650.5. Piconese S, et al. J Exp Med. 2008;205:825–839.6. Weinberg A. Cancer Res. 2013;73:7189–7198.
INCAGN1949 Demonstrates Increased Activation of Primary T Cells
INCAGN1949 Cooperates With Other Immunomodulatory Antibodies to Enhance T Cell Function
INCAGN1949 Mediates Effective OX40 Clustering
T Cell–Dependent Antibody Response (TDAR) in Cynomolgus Monkeys After Co-administration of
Vaccines and INCAGN1949
OX40 Is Selectively Expressed by Intratumoral Treg Cells in a Range of Primary Tumors
OX40-mCherry stably transfected Jurkat cells were co-cultured with plate-bound isotype control (A) or INCAGN1949 antibody (B). Arrows point to OX40 clustering. Confocal stack images are shown.
A. IL-2 secretion by human primary T cells in the presence of superantigen (Assay #1) and INCAGN1949 or isotype control (representative of n = 8 healthy donors).
B. Intracellular cytokine (IFNγ and TNFα) readout using flow cytometry post-anti-CD3 antibody stimulation (Assay #2) and plate bound INCAGN1949 or isotype control (representative of n = 10 healthy donors).
INCAGN1949 Is Well Tolerated In Vivo and Demonstrates a Linear PK Profile in Cynomolgus Monkeys
Mechanism 2: Anti-OX40 antibodies mediate the selective depletion of intratumoral regulatory T cells (Treg cell), thereby promoting anti-tumor activity(modified from ref. 2).
IL-2 secretion by human primaryT cells in the presence of superantigen (A–C) or anti-CD3 (D), and INCAGN1949 in combination with (A) anti-PD-1, (B) anti-PD-L1, (C) anti-CTLA-4,or (D) anti-GITR.
B
A
A B
B
IL-2 secretion by human primary T cells in the presence of superantigen and INCAGN1949 as compared to other anti-OX40 antibody variants (Ab1-Ab10) or isotype control (* significant P<0.05, Mann-Whitney test).
A
A A B
C DA
A. Th1 cytokines (IFNγ, GM-CSF, IL-2), Th2 cytokines (IL-6, IL-10), Th17 cytokines (IL-17) in plasma of cynomolgus monkeysadministered INCAGN1949 (0.5, 5, 20, and 100 mg/kg) in 5 weekly doses at day 22 post-inoculation (n = 4 monkeys per group).
B. Eight-week PK study of cynomolgus monkeys administered 5 weekly doses of INCAGN1949 at 0.5, 5, 20, and 100 mg/kg(n = 6–10 monkeys per group). From 3 different studies, plasma half-lives ranging from 8 to 15 days by either single or repeated administrations were calculated.
A. Representative histogram of OX40 expression from tumor-associated T effector cells or Treg cells (non-small cell lung carcinoma [NSCLC]).
B. Tabulated summary of OX40 expression by associated T effector cells (CD4+ T cells) and Treg cells.
C. Immunohistochemistry (IHC) of NSCLC sample stained for FoxP3+ Treg cells or an isotype control.
D. Intensity of OX40 or FoxP3 expression by IHC in a number of positive primary tumors (tumor tissue or tumor stroma tissue; n = 10 per tissue type).
Activity of Anti-OX40 Antibody Variants
Flow Cytometry IHC OX40 or FoxP3 Expression
OX40 Expression
INCAGN1949 Enhances Primary T Cell Function Across a Broad Range of Concentrations
10,000
0
IL-2
(pg/
mL)
8000
4000
2000
6000
*
IsotypeConfocal Stacks
3D
Top
Bottom
Top
Bottom
Bottom
Top
Top
Bottom
Bottom
Isotype
INCAGN1949
Assay #1IL-2 IFNγ TNFα
Assay #2
Antibody concentration (µg/mL)
0.01
3000
0
IL-2
(ng/
mL) 2000
1000
0.1 1 10 100 1000
Antibody concentration (µg/mL)
0.001
15
0
%C
D8+
IFNγ
T ce
lls
10
5
0.01 0.1 1 10 100
5
Antibody concentration (µg/mL)
0.001
15
0
%C
D4+
TNFα
T ce
lls
10
0.01 0.1 1 10 100
INCAGN1949Isotype
B C DAnti-PD-1
0
Donor 1
Donor 2
20 40 6010 30 50IL-2 in Supernatant (Fold Change)
Anti-PD-L1 Anti-CTLA-4 Anti-GITR
0
Donor 1
Donor 3
50 100 150
Donor 2
Donor 4
Immunomodulatory antibody INCAGN1949 Combination
Serum Cytokine Concentration
Indication Samples (n)
TeffCells
TregCells
NSCLC 5 +/− ++++Colorectal 4 +/− ++++Endometrial 3 +/− ++++Renal 3 — +++Breast 2 — ++Ovarian 2 — +Liver 2 +/− ++++Gallbladder 1 ++ +Pancreatic 1 +/− +Gastric 1 ++ ++++Head and neck 1 ++ ++++
NSCLC Breast Carcinoma
GastricCarcinoma
Controls0.5 mg/kg5 mg/kg20 mg/kg100 mg/kg
KLH-Specific B Cell Response (IgM)
ControlsINCAGN1949
TT-Specific T Cell Response
Days post-treatment
00
2500
SFU
/106
tota
l PB
MC
s
2000
1500
1 8 15 29 36
1000
43 57 66
500
TT-Specific B Cell Response (IgM)
0
500,000
0
µg/m
L
400,000
300,000
1 15 29
200,000
43 57 69
100,000
Days post-treatment
0
2000
0
µg/m
L
1500
1000
500
0
3000
0
µg/m
L
1 15 29
1000
43 57 69
2000
ControlsINCAGN1949
B PK Profile of INCAGN1949
107
101
INC
AGN
1949
con
cent
ratio
n (µ
g/m
L)
106
105
8 14 20 28
Days
104
103
102
0 2 4 6 10 12 16 18 22 24 26
0.5 mg/kg5 mg/kg20 mg/kg100 mg/kg
Intensity of Expression
10
0
Num
ber o
f tum
or
sam
ples
8
46
2
10
0
8
46
2
10
0
8
46
2
Tumor tissueTumor stroma OX40 FoxP3
Tumor tissueTumor stroma
NSCLC (FoxP3)FoxP3
Isotype
C D
1Agenus Inc., Lexington, MA; 2Agenus Switzerland Inc., Basel, Switzerland; 3Memorial Sloan Kettering Cancer Center, New York, NY; 4The Ludwig Institute for Cancer Research, New York, NY; 5Incyte Corporation, Wilmington, DE
Mechanism 1: Anti-OX40 antibodies mediate receptor forward signaling in the context of T cell antigen receptor (TCR) activation, enhance effector T cell activation, cytokine production, and survival, as well as promote memory T cell differentiation and reactivation (modified from ref. 1).
ActivationCD28
OX40co-stimulation
Memory generation
Clonalexpansion
Effector Contraction Reactivation
OX40co-stimulation
Naïve
Effector
Memory
NK CELL
Tregcell
INCAGN1949FcRs
Tregcell
MACROPHAGE
INCAGN1949FcRs
NK cellTreg cell
NeutrophilMacrophage
TUMOR
Treg cell depletion and effector T-cell co-stimulation
ADCC
ADCP
KLH-Specific B Cell Response (IgG)T effector cells Treg cells
OX40
INCAGN1949Isotype
INCAGN1949Isotype
Cynomolgus monkeys were vaccinated against tetanus toxoid (TT) or keyhole limpet hemocyanin (KLH)and administered 5 mg/kg INCAGN1949 or vehicle at day 1 and day 29; a vaccination booster was also given at day 29 (n = 6 monkeys per group). (A) TT-specific IFNγ T cell responses; (B) TT-specific IgM antibody responses; (C) KLH-specific IgM antibody responses; and (D) KLH-specific IgG antibody responses.
IL-17
IL-10
0
1000
0
750
40
24
500
20
250
2 6
pg/m
Lpg
/mL
0
1000
0
pg/m
L
750
40
24
500
20
250
2 6
IFNγ
IL-6
0
1000
0
400
24
800
200
600
2 6
GM-CSF
0
1000
0
750
40
24
500
20
250
2 6
IL-2
0
1000
0
750
40
24
500
20
250
2 6 0
1000
0
400
24
200
600
2 6
800
HoursHours
Donor 1
Donor 2
0 20 40 6010 30 50
Donor 1
Donor 2
0 20 40 6010 30 50
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